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Backbone

For eukaryotic
cell expression, we subcoloned cpVenus and cpCerulean into pEGFP vector with
EGFP removed beforehand, and with Nhe1 for cpCerulean and with NheI for cpVenus. pET-cpstFRET was created by connecting cpVenus to the C-terminal of cpCerulean in pET-52b(+) vector with BglII and SacI. Fore eukaryotic cell expression of cpstFRET, we subcloned it into pEGFP vector with NheI and ApaI. To create chimeric gene constructs of α-spectrin, we subcloned the gene encoding spectrin into pEGFP-C1 vector with AgeI and SacII from which the EGFP gene was removed beforehand. cpst-FRET was inserted into spectrin at amino acid position 1200, which located in the linker domain between the 10th and 11th spectrin repeat domains by restriction sites SalI and NotI.

Resource Information

A portion of this plasmid was derived from a plasmid made by

pEYFP-C1 Venus and pECFP-C1 Cerulean plasmids were generous gifts from David W. Piston, Vanderbilt University, Nashville, TN. Based on Venus and
Cerulean, we constructed circularly permutated cpVenus and cpCerulean. For
cpVenus, we first subcloned the gene fragments of 175Gly–239Lys from Venus
into pET-52b (+) vector with BamHI and EcoRI. Then we subcloned the gene fragments of 1Met–174Asp and connected
them to the C-terminal of the first fragment (supplementary material Fig. S1). Five
glycines were added between them to give flexibility with EcoRI and SacI. We generated cpCerulean similarly except that a
different primer was used for fragment 175Gly–239Lys, which was 59-
GGTACCAGGATCCATGGGCAGCGTGCAGC-39 with BamHI.

These plasmids were created by your colleagues. Please acknowledge the
Principal Investigator, cite the article in which the plasmids were described,
and include Addgene in the Materials and Methods of your future publications.